Module 2, Lab 05 - GAPDH Nested PCR

Thale cress, Arabidopsis thaliana

-----------------------------------------------------------------

Thursday (Round 1)

Today we started the exercise in which students will learn the basics of nested PCR. We will work with the gene that encodes one of the GAPDH isomers, GAPC, in the thale cress (Arabidopsis thaliana), the model organism of plants. Some people call it "the fruit-fly of plants".

GAPDH is an enzyme in charge of catalyzing one of the reactions in glycolysis. There are several nuclear genes that encode GAPDH isomers (proteins with different amino acid sequences but with the same function), and we are targeting the gene GAPC in the A. thaliana genome. We ran a first round of PCR, with our initial primers, and tomorrow we will run the second run, with the nested primers.

Friday (Round 2)

Today we ran the second round of PCR, using the nested primers.

In order to do so the first round primers were degraded adding an exonuclease to the amplified samples and incubating for 15 minutes at 37ºC. Then the exonuclease was denatured by incubating the samples at 80ºC for 15 minutes.

Finally, the Arabidopsis genomic DNA was diluted (1:50) and used to set up the second run of PCRs.

----------------------